Patterned expression of the
proneural genes ac and sc defines the PNCs for most external
sensory bristles in adult Drosophila, and ac-sc function is
required for PNC and SOP gene expression, as well as for specification of the
SOP cell fate. Fifteen of the genes identified by the combined cell
sorting/microarray approach also require proneural gene function for their
expression. In an ac− sc− proneural
mutant background, transcript accumulation from members of both the PNC
(CG11798, CG32434/loner, edl, PFE) and SOP
(CG3227, CG30492, CG32150, CG32392, Men,
qua) classes is lost from PNCs that require ac-sc function.
This result is further
evidence that the approach has identified bona fide PNC genes, and it
demonstrates that expression of these ten genes is, directly or indirectly,
downstream of the bHLH activators encoded by ac and sc. The data
further show that the PNC-specific imaginal disc expression of the previously
studied genes mira, phyl, rho, Spn43Aa, and Traf1 is likewise downstream of proneural gene function (Reeves, 2005).

The identification of sets of genes comprising the genetic programs deployed in
PNCs and SOPs by the action of proneural proteins offers a powerful opportunity
to investigate the regulatory organization of these programs. Specifically, it was of interest
to find out (1) which genes are directly activated by proneural
regulators, and which indirectly, and (2) the nature of the
cis-regulatory sequences and their cognate transcription factors that
distinguish PNC- versus SOP-specific target gene expression. This analysis was initiated
by examining potential regulatory sequences of several of the
genes that have been identified for the presence of conserved, high-affinity proneural
protein binding sites of the form RCAGSTG. The initial approach was to ask
whether evolutionarily conserved clusters of these binding sites identify
cis-regulatory modules of the appropriate specificity. To date, this
strategy has proven very successful. Genomic DNA fragments bearing
proneural protein binding site clusters associated with CG11798,
edl, Traf1, CG32434/loner, and rho confer
PNC-specific activity on a heterologous promoter,
while similar modules from CG32150, mira, and PFE drive
SOP-specific expression. In three cases, double
labeling with the SOP marker anti-Hindsight (Hnt) reveals that PNC-specific
expression of the reporter gene includes the SOP as well as the non-SOP cells.
Mutation of the proneural protein binding sites in four of the
enhancer-bearing fragments severely reduces (CG11798) or abolishes (CG32150,
edl, Traf1) reporter gene
expression in PNCs/SOPs. Such results indicate that these genes are indeed
direct targets of activation by proneural proteins in vivo (Reeves, 2005).

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).

To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).

Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).

Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).

Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).

A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).

The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).

Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).

In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).

The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.

The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).

Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).

To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).

To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).

In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).

Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).

The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).

The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).

Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).

S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).

To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).

Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).

Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).

Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).

Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).

Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).

The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).

It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).

Neuroblasts divide asymmetrically after delamination from Drosophila embryonic epithelium to generate a neuroblast and a smaller ganglion mother cell, by first forming an apical complex, which then targets cell fate determinants basally and reorients the mitotic spindle. This complex recruits Inscuteable, Partner of Inscuteable (Pins), and the G protein subunit Galphai to form an apical crescent at prophase. In metaphase, cell fate determinants and their respective adapters Numb/Partner of Numb, prospero mRNA/Staufen, and Prospero/Miranda form a basal crescent. Miranda is required for basal localization of Prospero, Staufen, and prospero mRNA, and binds not only Prospero and Staufen but also Inscuteable. Basal but not apical crescent formation requires the cortically localized Lethal giant larvae (Lgl) and Discs large (Dlg). Just how the apical complex directs basal crescent formation is not known. Because Miranda/Prospero and Staufen/prospero mRNA are transiently localized to the apical cortex before forming a basal crescent, conceivably Miranda is transported from the apical to the basal cortex by a motor, thereby mediating basal targeting of Prospero, Staufen, and prospero mRNA (Petritsch, 2002 and references therein).

What could be the motor for basal protein targeting? Lgl interacts with myosin II (Strand, 1994), and the lgl mutant phenotype is suppressed by a loss-of-function mutation of the myosin II gene zipper (zip). However, zip mutants exhibit no alterations in cell fate determinant localization or spindle orientation. Thus, motors other than Zip are probably involved in basal protein transport. To search for such motor proteins, Miranda-containing complexes were isolated from Drosophila embryos and two associated proteins were identified, myosin II/Zipper and myosin VI/Jaguar (Jar). Various methods were used to reduce jar activity and these treatments were found to disrupt Miranda localization and proper spindle orientation, but not the apical complex formation. As opposed to zip mutations, reducing jar activity enhances the basal protein transport defects in lgl mutants. Jar binds Miranda directly, and partially overlaps with Miranda in the distribution in neuroblasts, suggesting that Miranda and the complex it assembles including Prospero, Staufen, and prospero mRNA may be one of the cargos for Jar (Petritsch, 2002).

It thus appears likely that the hitherto unknown motor protein that mediates basal protein targeting may form a complex with Miranda. This possibility was tested and it was shown that Jar not only binds Miranda directly but is in a complex with Miranda in vivo. Also in a complex with Miranda is Zipper, a myosin II, which negatively regulates basal transport of Miranda. Jar exhibits a dynamic, punctate distribution concentrating at the basal side of the dividing neuroblast and partially overlapping when the Miranda basal crescent forms. Importantly, three independent ways of reducing Jar activity in neuroblasts all resulted in mislocalization of Miranda. In addition, a reduction or loss of Jar function compromised spindle orientation. However, the apical complex does not depend on Jar activity, suggesting that Jar acts downstream of or in parallel to the apical complex to ensure proper basal protein localization. These studies have therefore identified Jar as a myosin that targets basal proteins and aligns the mitotic spindle along the apical-basal axis in neuroblasts. The involvement of barbed end-directed myosins in asymmetric cell division has been demonstrated in budding yeast. In Drosophila neuroblasts, Jaguar, a pointed end-directed myosin motor, regulates asymmetric protein localization and spindle positioning (Petritsch, 2002).

It is intriguing that the same myosin VI may coordinate mitotic spindle alignment with the basal crescent of cell fate determinants. In an attempt to pursue this possibility further, an in vivo association of Jar with the microtubule-associated protein dEB1 has been demonstrated as well as an association of Jar with the microtubule-associated protein D-CLIP-190. However, loss-of-function mutations of dEB1 and D-CLIP-190 are not available. Reducing the levels of dEB1 by injection of double-stranded RNA (RNAi) results in a mild spindle orientation phenotype in epithelial cells, either due to functional redundancy -- there are four predicted dEB1 genes found in the database -- or due to a strong maternal contribution of dEB1. These technical difficulties have hampered attempts to examine the function of these genes in spindle orientation in neuroblasts (Petritsch, 2002).

It is not yet clear how Jar mediates basal targeting of Miranda. Identification of Jar as an essential motor protein for asymmetric division of neuroblasts raises the following questions for future studies: how might Jar interpret the apical-basal polarity set by the apical complex and target basal proteins? Does Jar organize actin cables along the apical-basal axis or simply move along preexisting actin filaments? Besides the pointed end-directed myosin VI, the barbed end-directed myosin II, Zip, is also associated with Miranda in vivo. Whereas both Jar and Lgl are required for basal protein targeting, loss of zipper function suppresses the lgl mutant phenotype. Given that Lgl and Jar synergize to control basal transport, Zip and Jar might have antagonistic activities in basal protein targeting. Genetic interactions between zipper and jar could not be easily assessed, due to overall abnormal morphology of the double mutant embryos at late stages. Whereas both Jar and Zip interact with Miranda in vivo, Zip is not detectable in Jar-containing complexes immunoprecipitated from embryo extracts, raising the possibility that Jar and Zip might compete for binding to Miranda (Petritsch, 2002).

In mitotic neuroblasts, the Prospero transcription factor and Numb, an antagonist of Notch
signaling, associate with their respective adapter proteins, Miranda and Partner of Numb
(Pon), and thereby localize to the basal cortex. In contrast, Inscuteable (Insc), Bazooka (Baz)
and Partner of Inscuteable (Pins) form a ternary complex at the apical cortex independently of
the basal determinants. However, the mechanisms that underlie the asymmetric protein
sorting in neuroblasts are not known. To address this issue, chromosomal
deficiencies have been sought that affect the subcellular distribution of Miranda. Such screening identified the
lgl tumor-suppressor gene that encodes a protein containing WD40 repeats. In
wild-type neuroblasts, Miranda, which localizes apically during interphase, accumulates at the
basal cortex upon mitosis after a transient spread into the cytoplasm. In germline
clone embryos lacking both maternal and zygotic lgl activity (lglGLC embryos), Miranda does
not localize asymmetrically in mitotic neuroblasts, but rather is distributed uniformly
throughout the cortex as well as in the cytoplasm, where it is concentrated along microtubule
structures. Consequently, Miranda segregates into both the daughter neuroblast
and the ganglion mother cell (GMC). Numb and Pon are also
distributed uniformly at the cortex and in the cytoplasm (Ohshiro, 2000).

Lgl is involved in epithelial polarity, and neuroblasts inherit Baz as an apical polarity cue
during the formation from neuroepithelia. In epithelia lacking lgl function, Baz
mislocalizes as irregular patches. However, Baz as well as Insc and Pins normally localize as
an apical crescent in the mutant neuroblasts, suggesting
that the apical cue is inherited by neuroblasts in the absence of lgl. Moreover,
neuroblast-specific expression of the lgl transgene can restore normal localization of
Miranda. Thus, Lgl probably functions autonomously within
neuroblasts for Miranda localization, and the phenotype in lgl neuroblasts would not result
from the inheritance of an abnormal polarity cue. Together, it is concluded that Lgl acts in the
localization of the basal determinants in neuroblasts, but not of the apical Baz-Insc-Pins
complex (Ohshiro, 2000).

Whether other tumor-suppressor genes contribute to protein localization
in neuroblasts was investigated. The tumor-suppressor gene dlg encodes a membrane-associated guanylate
kinase homolog. Germline clone embryos lacking both maternal and zygotic dlg activity
(dlgGLC embryos) exhibit defective localization of Miranda and Numb essentially identical to that of lglGLC embryos, suggesting that both
tumor-suppressor proteins function in the same process in neuroblasts. To investigate the
relationship between the roles of Dlg and Lgl, their subcellular localization in
neuroblasts was compared. Both Lgl and Dlg are distributed mainly throughout the cortex, whereas the
amount of Lgl in the cytoplasm appears to be greater in mitosis than in interphase.
The cortical localization of Lgl appears to be important for its function -- whereas the mutant
protein encoded by the temperature-sensitive allele lglts3 is distributed normally at
the permissive temperature (18°C), it fails to localize cortically at the restrictive temperature
(29°C). The wild-type Lgl protein exhibits a similar, abnormal
cytoplasmic distribution in dlgGLC embryos, whereas Dlg localization is not
affected in lglGLC embryos. Thus, the cortical localization of Lgl requires
dlg activity, suggesting that Dlg may function in localization of cell-fate determinants in
neuroblasts by positioning Lgl at the cortex (Ohshiro, 2000).

To distinguish whether Lgl establishes or maintains determinant localization, temperature-shift experiments were performed with the lglts3 allele. At 18°C, most (97%)
lgltsc/lgl minus embryos of females homozygous for lgltsc exhibit normal Miranda localization
at the basal cortex during metaphase and telophase. Ten minutes after shifting to
29°C, mislocalization of Miranda is apparent in 61% of metaphase or anaphase neuroblasts as well as in 43% of telophase neuroblasts, whereas the apical
localization of Miranda in interphase is not affected. On the basis of live
recordings of basal crescent formation in cells that express a fusion protein comprising
Miranda and green fluorescent protein, it is estimated that the time required for
neuroblast mitosis is 14.3+/-2.4 min. Thus, a temperature shift during
mitosis is able to induce mislocalization of Miranda, as is apparent in telophase neuroblasts,
indicating that Lgl functions during mitosis to determine Miranda localization. Metaphase arrest
can be induced in lglts3/lgl minus embryos by introduction of the fizzy (fzy) mutation. The
localization of Miranda in such metaphase-arrested neuroblasts is virtually insensitive to the
shift to 29°C. Thus, Miranda is not affected by the decrease in lgl function after it
has localized to the basal cortex. These results therefore indicate that Lgl may act early during
mitosis to recruit Miranda to the cortex but does not contribute to the maintenance of Miranda
in this location (Ohshiro, 2000).

Because Lgl is a component of cortical protein complexes that include nonmuscle Myosin II, or
Zipper (Zip), a test was performed for genetic interactions between lgl and zip in Miranda localization by
examining embryos zygotically mutant for both lgl and zip. The zip1 mutation does not affect Miranda localization throughout embryonic
development and lgl-zip embryos show no difference in Miranda localization from zygotic
lgl- embryos until late embryonic stages (stage 16) owing to the maternal contribution of zip. However, at
stage 17 when maternal zip had been exhausted, lgl-zip embryos appear to
restore the basal crescent of Miranda in metaphase neuroblasts, whereas
zygotic lgl- embryos at the same stage do not. Thus, Lgl might act
for Miranda localization in part by suppressing zip function directly or indirectly, consistent
with a study on yeast that indicated negative genetic interactions between Lgl homologs and
Myosin II. Alternatively, the asymmetric distribution of Pon requires myosin function in
neuroblasts, as revealed by the use of 2,3-butanedione monoxime (BDM) that generally
inhibits myosin function. The effect of BDM on Miranda localization was examined.
Treatment of wild-type embryos with BDM phenocopies lgl mutants, resulting in a partial
redistribution of Miranda from the cortex to microtubules. The effect of BDM is
more marked in lglGLC embryos: as the BDM concentration increases, the relocalization of
Miranda to microtubules is synergistically enhanced in most BDM-treated neuroblasts and
results in the complete exclusion of Miranda from the cortex at 50 mM BDM. The
phenocopy and enhancement of lgl mutations by general inhibition of myosin function are in
contrast with the suppressive effects of zip mutations, suggesting that Lgl cooperates with at
least one type of Myosin other than Zip to anchor Miranda at the cell cortex. It is thus inferred that
Lgl regulates negatively myosin II function and also positively the function of another Myosin
isotype in cortical protein targeting in neuroblasts (Ohshiro, 2000).

It would be expected that the abnormal distribution of Numb and Miranda in lgl mutant neuroblasts
results in incorrect determination of neural cell fate. Given the difficulty of monitoring neural cell
fate in severely distorted lglGLC embryos, this prediction was tested by analyzing the lineage of
the external sensory organ in the notum, in which all cell divisions are asymmetric and sibling
cells adopt distinct fates as a result of the asymmetric inheritance of Numb.
Sensory organ precursor cells in this lineage segregate Numb into a daughter cell pIIb, which
subsequently generates three inner cells (a glial cell, a neuron and a sheath cell). The sibling
pIIa cell divides into two outer cells constituting the external sensory structure, a hair and a
socket. Exposure of lglts3 mutant larvae to 29°C during external sensory organ development
mislocalizes Numb in mitotic precursor cells, as observed in neuroblasts, and
often transforms inner cells into outer cells resulting in duplicated external sensory structures,
a phenotype expected from loss of numb function. Indeed, this notum phenotype
is enhanced by reducing the numb gene dosage by half. Equal partition of Numb between sibling cells would result in numb gain
of function phenotypes because the half dose of numb is enough for correct cell-fate
decisions. The observed numb loss-of-function phenotype therefore suggests that a reduction
in lgl activity does not only equalize Numb distribution between sibling cells but also
attenuates numb function, consistent with the observation of cytoplasmic Numb in the lgl
mutants. Conversely, the presence of an extra numb gene induces opposite phenotypes under
the lgl mutant condition. The outer cells are frequently transformed into the
inner cells, resulting in the loss of the external sensory structure. This appearance of
the numb gain of function phenotypes is simply explained by the fact that the partition of
additional Numb from the transgene into both sibling cells raises numb activities over the
threshold necessary to suppress Notch function in both cells. These data thus indicate that Lgl is
essential in neural fate decisions through cortical targeting of cell-fate determinants (Ohshiro, 2000).

There are two important processes associated with the asymmetric division: (1) the
asymmetric localization of cell-fate determinants, which is achieved by specific adapter proteins
that themselves localize asymmetrically to the cortex in neuroblasts; and (2) the
orientation of the mitotic spindle and its coordination with the polarized localization of the
determinants, which requires the apical Baz-Insc-Pins complex. This study has revealed
another important process mediated by Dlg, Lgl and Myosins, which is responsible for the
cortical anchoring of the determinant-adapter complexes. This process occurs upstream of the
first and independently or parallel to the second of those two aspects of asymmetric division, as
the localization of Lgl and Dlg is independent of apical or basal components.
Both Lgl and Dlg contribute to the generation or maintenance of epithelial polarity, and zygotic mutants of the corresponding genes develop epithelial cell tumors as well as
brain tumors at late larval stages. These previous observations with epithelial cells,
together with the data on the roles of Lgl and Dlg in protein targeting in neuroblasts, suggest
that aberrant sorting of intracellular proteins may be responsible for the tumor formation
apparent in larval stages of lgl and dlg mutants (Ohshiro, 2000).

Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype; reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes (Rolls, 2003).

Both aPKC and Lgl are required for Miranda basal localization in neuroblasts, and all available data support a model in which Lgl is required for targeting Miranda to the neuroblast cortex, whereas aPKC blocks Lgl function on the apical side of the neuroblast: (1) lgl mutants have little or no Miranda at the cortex; (2) aPKC mutants show uniform cortical Miranda localization; (3) a weak lgl phenotype can be suppressed by reducing aPKC levels, showing that aPKC activity antagonizes Lgl activity; (4) aPKC and Lgl physically interact; (5) an overexpressed nonphosphorylatable Lgl protein is uniformly cortical and able to induce uniform cortical Miranda localization, whereas phospho-Lgl is preferentially released from the cell cortex. This has led to a model in which Lgl acts as an anchor for Miranda at the basal cortex, but is absent from the apical cortex due to aPKC-mediated phosphorylation. Although this simple model is attractive, it is noted that Lgl has never been observed colocalized with Miranda in a basal cortical crescent, and a role for cytoplasmic Lgl in Miranda localization has not been definitively ruled out (Rolls, 2003).

Domain mapping of Scrib reveals a multistep localization mechanism and domains necessary for establishing cortical polarity: the LRR domain of Scrib is sufficient to target Miranda protein to the neuroblast cortex, but LRR+PDZ will exclude Miranda from the cortex

The Drosophila tumor suppressor protein Scribble is required for epithelial polarity, neuroblast polarity, neuroblast spindle asymmetry and limiting cell proliferation. It is a member of the newly described LAP protein family, containing 16 leucine rich repeats (LRRs), four PDZ domains and an extensive carboxyl-terminal (CT) domain. LRR and PDZ domains mediate protein-protein interactions, but little is know about their function within LAP family proteins. This study has determined the role of the LRR, PDZ and CT domains for Scribble localization in neuroblasts and epithelia, and for Scribble function in neuroblasts. It was found that the LRR and PDZ domains are both required for proper targeting of Scribble to septate junctions in epithelia; that the LRR domain is necessary and sufficient for cortical localization in mitotic neuroblasts, and that the PDZ2 domain is required for efficient cortical and apical localization of Scribble in neuroblasts. In addition, it is shown that the LRR domain is sufficient to target Miranda protein to the neuroblast cortex, but that LRR+PDZ will exclude Miranda from the cortex. These results highlight the importance of both LRR and PDZ domains for the proper localization and function of Scribble in neuroblasts (Albertson, 2004).

In the absence of all Scrib function, Mira is predominantly localized to the cytoplasm and mitotic spindle of neuroblasts. Expression of just the Scrib LRR domain results in uniform cortical Scrib LRR distribution and the restoration of uniform cortical Mira localization. Conversely, all Scrib proteins that lack the LRR domain (PDZ, DeltaLRR and CT) fail to efficiently target Mira to the cortex. These results reveal a positive role for the Scrib LRR domain in targeting Mira to all regions of the neuroblast cortex. A similar 'uniform cortical Mira' phenotype is also observed in certain aPKC and lgl genetic backgrounds. Neuroblasts lacking aPKC show uniform cortical Mira and neuroblasts misexpressing a dephospho-Lgl protein also show uniform cortical Mira. In addition, loss of lgl leads to cytoplasmic Mira localization in neuroblasts. This has led to a model in which the apically-localized aPKC phosphorylates Lgl to inactivate it, thus restricting active dephospho-Lgl to the basal cortex, where it promotes cortical localization of Mira. The Scrib LRRs could act upstream of aPKC and Lgl, perhaps by blocking aPKC/Lgl interactions, and thus allowing activated Lgl to target Mira to the entire cortex. Alternatively, the Scrib LRRs could act downstream of aPKC and Lgl, perhaps by allowing both dephospho- and phospho-Lgl to target Mira to the cortex. In addition, loss of jaguar (myosin VI) leads to cytoplasmic localization of Mira, raising the possibility that the Scrib LRRs could stimulate myosin VI activity around the neuroblast cortex to promote uniform cortical Mira localization. The identification of Scrib LRR-binding proteins will help distinguish between these models (Albertson, 2004).

Addition of the PDZ domains back to the Scrib LRR protein dramatically alters the function of the protein. Whereas the Scrib LRR protein is uniformly cortical and promotes Mira cortical localization, Scrib LRR+PDZ proteins (FL, DeltaCT) are apically enriched and exclude Mira from the apical cortex. Thus, addition of the PDZ domains switches Scrib from promoting cortical Mira localization to excluding cortical Mira localization. The PDZ domains could carry out this function of excluding Mira from the apical cortex in at least three different ways. (1) The Scrib PDZ domains could promote aPKC-Lgl interactions, thereby leading to the phosphorylation and inactivation of apical Lgl; this would restrict active Lgl to the basal cortex, where it promotes cortical Mira localization. (2) The Scrib PDZ domains could promote myosin II (zipper) activity at the apical cortex; myosin II is a known inhibitor of Lgl, and thus this would restrict active Lgl to the basal cortex where it could promote cortical Mira localization. (3) The Scrib PDZ domains could provide directionality to the actin-myosin VI cytoskeleton, which could transport Mira specifically to the basal cortex. Identification of proteins that interact with the Scrib PDZ domains would help distinguish between these models (Albertson, 2004).

Little is known about how Dlg, Scrib and Lgl regulate cell size asymmetry and spindle asymmetry. LRR and PDZ domains of Scrib are necessary for this function. How might Scrib regulate cell size and spindle asymmetry? Two good candidate effectors are Ran GTPase and Pins. LRRs are known to physically interact with Ran, which promotes spindle assembly through several target proteins. For example, Ran stimulates the activity of NuMA (a microtubule motor accessory protein that promotes spindle assembly) by destabilizing inhibitory complexes associated with NuMA. LGN (a mammalian Pins homolog) is essential for mitotic spindle assembly and binds NuMA; release from LGN is an important event in the activation of mitotic NuMA. In Drosophila, Pins physically interacts with Dlg and is asymmetrically localized to the apical cortex of mitotic neuroblasts, where it promotes spindle asymmetry. These data suggest possible links between Dlg, Scrib, Ran and Pins and establishment of mitotic spindle asymmetry. Genetic and biochemical studies investigating interactions between Scrib, Ran and Pins may further understanding of spindle asymmetry establishment in Drosophila neuroblasts (Albertson, 2004).

Deletion of the CT domain has no effect on Scrib localization or its ability to rescue all tested scrib mutant phenotypes in neuroblasts; the CT domain alone is cytoplasmic and has no rescuing ability in any assay performed. It is concluded that the CT domain is not essential for any aspect of Scrib localization or function tested here (Albertson, 2004).

scrib transgenic lines that specifically lack the LAPSDa/b domains have not been assayed, however, some conclusions can be drawn based on existing Scrib domain analysis. The Scrib DeltaPDZ protein contains both LAPSDa/b domains, is membrane targeted but not enriched apically; it fails to promote basal Mira targeting, and it is defective for asymmetric mitotic spindle and cell size asymmetry. Thus, the LAPSD domains are insufficient for apical enrichment of Scrib and all of its tested functions in neuroblasts. The Scrib LRR protein lacking the LAPSDb domain is still membrane-associated, showing that the LAPSDb domain is not required for Scrib membrane targeting. Some evidence was found that the LAPSDb domain regulates nuclear import/export of the Scrib protein. The LRR protein contains just the LRRs and the LAPSDa domain and is targeted to the nucleus; this shows that there is a nuclear import signal or binding site for a nuclear protein within the LRR/LAPSDa domains, although a predicted nuclear localization signal is not detectable within these domains. In contrast, the DeltaPDZ protein contains the same LRR/LAPSDa domains plus the LAPSDb and CT domains, and it is excluded from the nucleus. This shows that the LAPSDb or CT domains can prevent nuclear import of the LRR/LAPSDa protein; it is highly likely that this function is provided by the LAPSDb domain, because deletion of the CT domain from an otherwise wild-type Scrib protein (i.e., DeltaCT) does not result in nuclear localization. These results are in contrast to the role of the LAPSDa/b domains in the related C. elegans Let-413 protein, where the LAPSDa/b domains are required for establishing epithelial polarity but not Let-413 protein localization. It will be interesting to determine whether the Scrib LAPSDa/b domains play a similar role in Scrib epithelial localization (Albertson, 2004).

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC 'buds' off. This compensatory mechanism has been referred to as 'telophase rescue'. The Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (TRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. TRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires TRAF1. These data suggest that TRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway (Wang, 2006).

In telophase NBs, segregation of cell fate determinants, such as Pros, into future GMCs, is critical for their proper development. Telophase rescue appears to be one of the safeguard mechanisms that acts to ensure that GMCs inherit the cell fate determinants and adopt the correct cell identity when the mechanisms, which normally operate during NB divisions, fail (e.g., in insc mutant). Telophase rescue is a phenomenon for which the underlying mechanism involved remains largely unknown. The current data demonstrate that TRAF1 and Egr are two members of the Insc-independent telophase rescue pathway specific for Mira/Pros (Wang, 2006).

Although it is apically enriched in mitotic NBs and can directly interact with Baz in vitro, TRAF1 does not seem to be involved with the functions normally associated with the apical complex proteins. One distinct feature of TRAF1 differs from the other known apical proteins is its localization pattern; it is cytoplasmic in interphase and the apical crescent is prominent only at metaphase. In contrast, proteins of the apical complex are largely undetectable during interphase and form distinct apical crescents, starting from late interphase or early prophase. The protein localization difference between TRAF1 and other apical proteins suggests that TRAF1 and apical proteins are not always colocalized during mitosis. If TRAF1 is a bona fide member of the apical complex, the localization defects of other apical proteins are expected to be observed in TRAF1 mutant, as well as mislocalization of basal proteins, which was not detect. In addition, no spindle orientation or geometry defects were observed in the absence of TRAF1. Based on these observations, it is concluded that TRAF1 is not involved with the functions normally associated with the apical complex proteins (Wang, 2006).

The in vitro GST fusion protein pull-down assay suggests that TRAF1 may physically bind to Baz. This result is consistent with genetic data, indicating that TRAF1 acts downstream of baz and that its apical localization requires baz. These observations are consistent with the view that TRAF1 is recruited to the apical cortex by apical Baz in mitotic NBs. Baz, even at very low levels, can recruit TRAF1 to the apical cortex of the mitotic NBs. For example, in insc mutant NBs, TRAF1 remains apical probably owing to the low levels of Baz that remain localized to the apical cortex. This speculation is supported by Mira/Pros telophase rescue data, which clearly demonstrate that the telophase rescue seen in insc mutant NBs is severely damaged in baz mutant, suggesting that the Baz function required for Mira/Pros (and Pon/Numb) telophase rescue is intact in insc mutant NBs (Wang, 2006).

It has been shown that Pins/Gαi asymmetric cortical localization can be induced at metaphase by the combination of astral microtubules, kinesin Khc-73 and Dlg in the absence of Insc; this coincides with the observation that TRAF1 also forms tight crescent only at metaphase in both WT and insc mutant NBs. Does TRAF1 apical crescent formation also require the functions of astral microtubules, kinesin Khc-73 and Dlg? The data do not favor this hypothesis based on the following observations. (1) In TE35BC-3, a small deficiency uncovering sna family genes insc is not expressed but Pins and Gαi are asymmetrically localized, indicating that the astral microtubules, kinesin Khc-73 and Dlg pathway remain functional. TRAF1 is delocalized and is uniformly cortical in this deficiency line. (2) Similarly, in egr insc NBs, TRAF1 is cytoplasmic whereas the functions of astral microtubules, kinesin Khc-73 and Dlg are intact. (3) In egr NBs TRAF1 is cytoplasmic, whereas the apical complex is normal and astral microtubules, kinesin Khc-73 and Dlg are present. (4) TRAF1 apical localization remains unchanged in dlg mutant NBs. Based on these observations, it is concluded that TRAF1 apical localization is unlikely to share similar mechanism with Pins and Gαi and is likely to be independent of astral microtubules, kinesin Khc-73 and Dlg. TRAF1 apical localization appears to specifically require Egr and Baz (Wang, 2006).

In TRAF1 insc double-mutant embryos, the complete segregation of Mir/Pros into future GMCs occurs only in about 12% of the total population, and in the remaining NBs, only a fraction of Mira/Pros segregate into future GMCs as indicated by the Mira 'tail' extending into the future NBs at telophase. As it is difficult to address the global effect of this partial segregation of Mira/Pros on GMC specification in TRAF1 insc double mutant, focus was place on a well-defined GMC, GMC4-2a in NB4-2 lineage, to evaluate this issue. It is assumed that as long as the RP2 neuron (progeny of GMC4-2a, Even-skipped (Eve)-positive) was identified in a particular hemisegment, the GMC cell fate of GMC4-2a in that hemisegment should have been correctly specified. In insc mutants, almost all hemisegments contain RP2s, indicating that GMC4-2a has adopted the correct GMC cell fate in 99% of the total hemisegments. When TRAF1 insc double-mutant embryos were stained with anti-Eve, it was found the frequency of loss of Eve-positive RP2 neuron increased (to 8%) in late embryos, suggesting that about 8% of the GMCs in TRAF1 insc double mutant did not inherit sufficient Pros to specify the GMC fate in these embryos. The relatively low frequency (8%) of mis-specification of GMCs suggests that the threshold amount of Pros protein needed is sufficiently low such that just a partial inheritance of Pros, even when telophase rescue is compromised, is sufficient for most GMCs to be correctly specified (Wang, 2006).

Although Mira/Pros and Pon/Numb share similar basal localization patterns in insc NBs, further removal of either TRAF1 or Egr compromised telophase rescue only for Mira/Pros, but not for Pon/Numb. This difference between Mira/Pros and Pon/Numb indicates that the detailed mechanisms of basal localization and segregation of Mira/Pros differ from those of Pon/Numb, which is consistent with the observations that the dynamics of Mira/Pros and Pon/Numb localization early in mitosis are different and the basal localization for Mira/Pros and Pon/Numb requires different regions of the Insc coding sequence (Wang, 2006).

Dlg/Lgl/Scrib are required for correct basal localization of Mira/Pros and Pon/Numb in mitotic NBs. Dlg has been shown to be involved in the Mira telophase rescue. In dlg insc double-mutant NBs, not only was spindle geometry symmetric but Mira telophase rescue was also affected. It would be interesting to know if Dlg belongs to the same pathway as TRAF1 and Egr and if Dlg is also involved in Pon/Numb telophase rescue (Wang, 2006).

Two other members of the TRAF family have also been identified in Drosophila: DTRAF2 (DTRAF6) and DTRAF3. In contrast to the specific and strong expression of TRAF1 in the embryonic NBs, only low levels of ubiquitous signals similar to the control background were seen in the NBs with DTRAF2 and DTRAF3 probes. It is likely that DTRAF2 and DTRAF3 are not expressed in NBs and do not play an important role in Mira/Pros telophase rescue pathway as the Mira/Pros telophase rescue is dramatically compromised in TRAF1 insc and egr insc NBs (Wang, 2006).

In mammals, the TNF pathway works as a typical receptor-mediated signal transduction pathway. TNFR is a key player in transducing external signal to the cytoplasm. In the Drosophila compound eyes, ectopic Egr, Wgn and TRAF1 seem to work in a similar receptor-mediated signal pathway to induce apoptosis through the activation of the JNK pathway. Does the same Egr, Wgn and TRAF1 receptor-mediated signal pathway play a role in Mira/Pros telophase rescue? If it does, the coexpression of Egr, Wgn and TRAF1 might be expected to be seen in dividing NBs, along with the potential interaction between TRAF1 and the cytoplasmic domain of Wgn. Three observations argue against this hypothesis: (1) wgn is not expressed in embryonic NBs but in the mesoderm. (2) The domain analysis suggests that the Drosophila Wgn cytoplasmic domain is unique with no sequence homology to any mammalian TNFR family members and has neither a TRAF-binding domain nor a death domain, which is required for the interaction between TNFR and TRAF in mammals. (3) More informatively, Wgn knockdown by a UAS head-to-head inverted repeat construct of wgn (UAS-wgn-IR) driven by a strong maternal driver, mata-gal4 V32A, in WT embryos did not affect TRAF1 apical localization. These observations are consistent with the view that the receptor Wgn may not be involved in Mira/Pros telophase rescue or is redundant in this pathway. If this is the case, then how do TRAF1 and Egr function in Mira/Pros telophase rescue? It has been reported that TRAFs associate with numerous receptors other than the TNFR superfamily in mammals. It is speculated that Egr and TRAF1 may adopt an alternative receptor in NBs for Mira/Pros telophase rescue. However, until an anti-Wgn antibody and wgn mutant alleles are available, the possibility that Wgn is involved in Mira/Pros telophase rescue cannot be ruled out (Wang, 2006).

In embryos deficient for miranda, Prospero is not associated with the membrane, but stays in the cytoplasm in prophase. Prospero remains in the cytoplasm in metaphase and anaphase and then is segregated into both daughter cells. Shortly after cell division, Prospero is translocated into the nuclei of both daughter cells. The orientation of the mitotic spindle in neuroblasts and cells of the procephalic neurogenic region is normal in miranda deficient embryos. A transduced miranda can rescue the Prospero localization defects in neuroblasts of miranda indicating that miranda is required for the correct positioning of Prospero in neuroblasts during mitosis (Shen, 1997).

The Numb protein is asymmetrically localized to the basal cell membrane in neuroblasts and cells of the procephalic neurogenic region during mitosis, in contrast to the cytoplasmic distributions of Prospero. After cell division, Numb is segregated into only the basal daughter cell, whereas Prospero is translocated into the nuclei of both cells (Shen, 1997).

The asymmetric localization of Miranda in neuroblasts of prospero and numb mutants is indistinguishable from that of wild-type embryos. Therefore, the asymmetric localization of Miranda does not require prospero or numb. In embryos homozygous for a null allele of inscuteable, both Miranda and Prospero are unable to form crescents or they form crescents that are randomly localized along the cell membrane. Therefore Miranda crescent formation and localization requires inscuteable (Shen, 1997).

In the GMC, Prospero translocates to the nucleus, where it establishes differential gene expression between sibling cells. miranda, which encodes a new protein that co-localizes with Prospero in mitotic neuroblasts, tethers Prospero to the basal cortex of mitotic neuroblasts, directing Prospero into the GMC, and releases Prospero from the cell cortex within GMCs. miranda thus creates intrinsic differences between sibling cells by mediating the asymmetric segregation of a transcription factor into only one daughter cell during neural stem-cell division. The expression of even-skipped was followed in embryos mutant for six miranda alleles. A stereotyped pattern of GMCs and neurons express eve. The well characterized aCC/pCC, RP2, CQ, U, and EL neurons all express eve. In prospero mutant embryos, the aCC/pCC and RP2 neurons fail to express eve and most U and CQ neurons also fail to express eve. It was expected that all miranda alleles would show reduced Prospero activity in the GMC either because Prospero inappropriately segregates into both neuroblasts and GMCs, or because Prospero fails to translocate efficiently into the nuclei of GMCs. This predicts that the EVE CNS phenotype of miranda mutant embryos might resemble the Eve CNS phenotype of prospero mutant embryos, should miranda exert its effect through its ability to bind, segregate and release Prospero. This is the case for two catagories of miranda mutants. For the five alleles in which Prospero falls off the cortex (the inner surface of the cell membrane) of neuroblasts, there is an observed reduction of about one-half, in the number of RP2, aCC/pCC, U and CQ neurons expressing eve. Consistent with a decrease in the level of Prospero protein distributed into GMCs, this phenotype resembles a weak prospero phenotype. A one-half reduction in the number of eve-expressing EL neurons is observed; in prospero mutants all EL neurons form normally. This additional eve phenotype may result from the ectopic expression of Pros in neuroblasts or from defects in the partition of other factors dependent on Miranda function. This study raises some interesting questions. Miranda is itself asymmetrically localized: (1) what proteins tether it to the basal cortex of neuroblasts? (2) What proteins regulate miranda so that it releases Prospero in the GMC once cytokinesis is complete? (Ikeshima-Kataoka, 1997).

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, Prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen. Staufen interacts in vivo with a segment of the Prospero 3' UTR. To assay RNA binding in vivo, the Prospero 3' UTR was injected into embryos expressing a green fluorescent protein (GFP)-Staufen protein fusion and the formation of Staufen ribonucleoprotein particles (RNP) was monitored. The full-length Prospero 3' UTR forms particles, as does the Bicoid 3' UTR, but not the coding region of the Prospero mRNA even though it is able to form an extended secondary structure. These RNPs are associated with the nuclei of the precellular embryo, and move with them to the cortex at stage 4. However, unlike the RNP particles formed between Staufen and the Bcd 3' UTR, the Staufen/Prospero 3' UTR particles do not associate with the astral microtubules. Similar results are observed when the Prospero 3' UTR is injected into embryos expressing wild-type Staufen (detected with anti-Staufen antibodies), rather than a GFP fusion. To further map the region of the Prospero 3' UTR with which Staufen interacts, either the 3' half of the UTR, or the 5' half were injected into embryos. Whereas the 3' segment recruits Staufen into RNPs within 5-10 min of injection, the 5' segment does so only slightly, if at all, after 20-30 min. Therefore, the region of the Prospero mRNA recognized by Staufen lies in the terminal 650 bases of the mRNA (Schuldt, 1998).

Staufen colocalizes with Prospero protein at all stages of the cell cycle. In embryos, Staufen is concentrated on the apical side of the neuroblast at interphase, then forms a crescent on the basal side of the cell in prophase, where it remains through mitosis before partitioning to the GMC at division. A similar subcellular distribution is seen in living embryos. This dynamic pattern of localization shows Staufen to be correctly placed to bind the Prospero mRNA throughout the cell cycle, and to mediate its segregation into the GMC (Schuldt, 1998)

Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein. Experiments in the oocyte have identified two regions of Staufen that are required for its function during oogenesis: a 99-amino-acid region in the middle of dRBD2, and the carboxy-terminal 157 amino acids that include dRBD5. As neither the dRBD2 insert nor the carboxy-terminal domain binds dsRNA in vitro, these two regions may be involved in some other aspect of Staufen function. When Staufen protein that lacks either the dRBD2 insert or the carboxy-terminal domain is expressed maternally, it can only partially rescue the abdominal defects caused by Staufen null mutations To test whether either of these domains is required for Staufen localization in neuroblasts, the subcellular distribution of Staufen mutants that lack the dRBD2 insert (dRBD2) or the carboxy-terminal domain of Staufen (dRBD5) were assayed. The removal of the dRBD2 insert has no effect on Staufen distribution in neuroblasts at any stage of the cell cycle. However, the loss of the carboxy-terminal 157 amino acids of Staufen eliminates both apical and basal localization. Staufen dRBD5 is distributed throughout the cytoplasm from interphase through mitosis. Therefore, the carboxyl terminus of Staufen is necessary to direct asymmetric distribution of the protein in neuroblasts. If the normal subcellular distribution of Staufen is mediated by a specific protein-protein interaction, then the site of the interaction may reside within the 157-amino-acid domain removed in Staufen dRBD5 (Schuldt, 1998).

Staufen binds directly to Miranda protein via the Staufen dRBD5 motif. Staufen dRBD5 does not interact with RNA in vitro but is required in vivo for Staufen protein localization, suggesting that it may interact with other proteins that anchor Staufen at the apical and basal cortex, or mediate Staufen's transport from one side of the neuroblast to the other. To identify proteins that might interact with dRBD5 to direct Staufen crescent formation, a yeast two-hybrid screen was carried out on a random-primed embryonic cDNA library using a LexA-dRBD5 fusion protein as bait. From 4,000,000 transformants, 10 positive clones were isolated, and the library plasmid could be recovered from 6 of these. All six clones contain the same insert, which encodes amino acids 506-776 of the Miranda protein. To test the specificity of the dRBD5/Miranda interaction, the Miranda clone was retransformed into yeast that contained either the original bait (lexA-dRBD5) or the control baits (lexA-lamin or lexA-BRCA2). Only yeast containing both lexA-dRBD5 and Miranda-VP16 expresses high levels of beta-galactosidase. Furthermore, this region of Miranda does not interact with all dRBDs, because no beta-galactosidase activity is observed when Miranda-VP16 is cotransformed with LexA-Staufen dRBD1. Thus, Miranda binds specifically to Staufen dRBD5, and does not interact with a closely related domain from the same protein (Schuldt, 1998).

To more precisely map the region of Miranda protein that interacts with Staufen-dRBD5, the fragment of Miranda identified in the yeast two-hybrid screen was divided into two parts (amino acids 506-638 and amino acids 639-776), and their interaction with dRBD5 was examined in a GST pull-down assay. 35S-labeled Staufen-dRBD5 coprecipitates with both the full-length fragment, GST-Miranda amino acids 506-776, and the amino-terminal segment of this region, GST-Miranda amino acids 506-638, but shows no interaction above background with the carboxy-terminal segment, GST-Miranda amino acids 639-776. This suggests that the Staufen binding site in Miranda corresponds to the predicted coiled-coil domain that extends from amino acids 526-593 (Schuldt, 1998).

Miranda colocalizes with Staufen protein and Prospero mRNA during neuroblast divisions, and neither Staufen nor Prospero RNA are localized in miranda mutants. Like Staufen, Miranda concentrates predominantly on the apical side of the cell at interphase. Interestingly, Miranda mRNA is also localized predominantly on the apical side of the neuroblast. Miranda protein then forms a crescent on the basal side of the neuroblast at prophase, where it remains until after cell division. Therefore, the subcellular distribution of Miranda suggests that it might interact with Staufen at all stages of the cell cycle (Schuldt, 1998).

It is concluded that Miranda binds to Prospero protein and to Staufen, which in turn binds Prospero mRNA, to form a complex on the apical side of the neuroblast. The complex may be anchored by Inscuteable at interphase, and then released as the cell cycle progresses. In mirandaRR127, Staufen accumulates on the apical side of the cell, suggesting that Miranda may regulate release from the apical cortex. Miranda, Prospero, Staufen, and Prospero mRNA then move as a group to the basal side of the cell during mitosis, a process that appears to require actin microfilaments. Staufen and Miranda also associate with the apical centrosome, although the significance of this interaction is unclear. Once at the basal cortex, the complex is anchored by factors that have not, as yet, been identified. However, as Miranda acts as the adapter between protein and RNA localization, these factors may be isolated in screens for other Miranda binding proteins. After cytokinesis, Miranda is rapidly degraded in the GMC, and Prospero is released and enters the nucleus. It may be important, therefore, to minimize translation of new Miranda protein in the GMC. Whereas Prospero mRNA is specifically segregated to the GMC, Miranda mRNA remains tightly anchored on the apical side of the neuroblast. By tethering Miranda mRNA in this way, Miranda protein, but not Miranda mRNA, is partitioned to the GMC at cell division (Schuldt, 1998).

Several interesting questions remain to be answered. What regulates the release of Miranda from the apical side of the cell? How are Miranda, Prospero, Staufen, and Prospero mRNA transported to the basal side of the neuroblast? Do they move as a complex, and how are they anchored at the basal cortex? Prospero and Staufen bind to the same region of Miranda, but it is not known whether they bind to the same molecule simultaneously. The answers to these questions may help to elucidate the mechanism of asymmetric protein and RNA localization not only in the nervous system, but also in other tissues, and in other organisms (Schuldt, 1998).

An important question in cellular and developmental biology is how a cell divides to produce daughter cells with different fates. Drosophila neuroblasts are a model
system for studying asymmetric cell division: at each division, neuroblasts retain stem cell-like features, whereas their sibling ganglion mother cell (GMC) has a more
restricted fate. Establishing neuroblast/GMC differences involves the asymmetric localization of proteins (Inscuteable, Miranda, Prospero, and Staufen) and RNA
(Prospero). All of these factors are apically localized during interphase, and all except Inscuteable move to the basal cortex at mitosis prior to being partitioned solely
into the GMC. Miranda is colocalized with Staufen and Prospero in neuroblasts, and is required for the asymmetric cortical localization of
both proteins. Analysis of miranda mutants reveals three functional domains within the Miranda protein: (1) an N-terminal domain (1-290 aa) sufficient for
association of Miranda with the cell cortex and basal localization in mitotic neuroblasts; (2) a central domain (446-727 aa) necessary for apical localization in
interphase neuroblasts as well as for 'cargo binding' of Prospero, Staufen, and Prospero mRNA, and (3) a C-terminal domain (727-830 aa) necessary for the timely
degradation of Miranda and release of its cargo from the cortex of the newborn GMC. In addition, Miranda is asymmetrically localized in epithelial cells that lack
Inscuteable and divide symmetrically; thus the mechanism regulating Miranda localization is common to epithelial cells and neuroblasts, and Inscuteable is not an
obligate component. A C-terminal domain of Staufen is defined that is sufficient for Miranda-dependent cortical localization in neuroblasts (Fuerstenberg, 1998).

Cellular diversity in the Drosophila central nervous system is generated through a series of asymmetric cell divisions in which one progenitor produces two daughter
cells with distinct fates. Asymmetric basal cortical localization and segregation of the determinant Prospero during neuroblast cell divisions play a crucial role in
effecting distinct cell fates for the progeny sibling neuroblast and ganglion mother cell. Similarly asymmetric localization and segregation of the determinant Numb
during ganglion mother cell divisions ensures that the progeny sibling neurons attain distinct fates. The most upstream component identified so far which acts to
organize both neuroblast and ganglion mother cell asymmetric divisions, is encoded by inscuteable. The Inscuteable protein is itself asymmetrically localized to the
apical cell cortex and is required both for the basal localization of the cell fate determinants during mitosis and for the orientation of the mitotic spindle along the
apical/basal axis. The functional domains of Inscuteable have been defined. Amino acids 252-578 appear sufficient to effect all aspects of its function, however, the
precise requirements for its various functions differ. The region aa288-497 is necessary and sufficient for apical cortical localization and for mitotic spindle
(re)orientation along the apical/basal axis. A larger region (aa288-540) is necessary and sufficient for asymmetric Numb localization and segregation; however, correct
localization of Miranda and Prospero requires additional sequences from aa540-578. The requirement for the resolution of distinct sibling neuronal fates appears to
coincide with the region necessary and sufficient for Numb localization (aa288-540). These data suggest that apical localization of the Inscuteable protein is a
necessary prerequisite for all other aspects of its function. Although Inscuteable RNA is normally apically localized, RNA localization is not
required for protein localization or any aspects of inscuteable function (Tio, 1999).

Neuroblasts in the developing Drosophila CNS asymmetrically localize the cell fate determinants Numb and Prospero as well as Prospero RNA to the basal cortex during mitosis. The localization of Miranda to the apical cortex, its interaction with Inscuteable in vitro and its role in localizing several downstream factors suggests that Miranda occupies a central link between Inscuteable at the apical cortex and the localization of Prospero, Staufen, and Prospero RNA to the basal cortex. How, early in mitosis, the apically localized Inscuteable dictates basal localization of intrinsic factors for asymmetric cell division may be elucidated by further studies on the genetic and cell biological mechanisms of the asymmetric localization of Miranda. The localization of Prospero requires the function of inscuteable and miranda, whereas Prospero RNA localization requires inscuteable and staufen function (Shen, 1998).

Miranda forms a crescent on the apical cortex of neuroblasts in late interphase. Later in mitosis, Miranda forms a crescent on the basal neuroblast cortex. Asymmetric localization of both Numb and Prospero has been shown to be dependent on the actin cytoskeleton. The actin dependence of Miranda localization was tested using the actin depolymerizing drug latrunculin A. After treatment of Drosophila embryos with 200 µM latrunculin A for 20 min, asymmetric localization of Miranda is completely disrupted, while membrane association is unperturbed. It is concluded that the asymmetric localization of Miranda during mitosis is an actin-dependent process. All Miranda fragments that contain the amino-terminal 298 amino acids exhibit the same asymmetric localization pattern as wild type Miranda. In contrast, a fragment containing amino acids 114-298 localizes to the cytoplasm and fails to segregate preferentially into the basal daughter cell, as does a fragment containing all residues carboxy-terminal to amino acid 300 (Shen, 1998).

The observation that Miranda protein fragments form an apical crescent that may coincide with the apical Inscuteable crescent led to a test of the possibility that Miranda interacts physically with Inscuteable. In an in vitro binding assay, Inscuteable coprecipitates with Miranda. An Inscuteable fragment from amino acids 252 to 615 also interacts with Miranda (Shen, 1998).

Miranda contains multiple functional domains: an amino-terminal asymmetric localization domain, which interacts with Inscuteable; a central Numb interaction domain, and a more carboxy-terminal Prospero interaction domain. Miranda and Staufen have similar subcellular localization patterns and interact in vitro. miranda function is required for the asymmetric localization of Staufen. Miranda localization is disrupted by the microfilament disrupting agent latrunculin A. These results suggest that Miranda directs the basal cortical localization of multiple molecules, including Staufen and Prospero mRNA, in mitotic neuroblasts in an actin-dependent manner (Shen, 1998).

When neuroblasts divide, Prospero protein and Pros mRNA
segregate asymmetrically into the daughter neuroblast and
sibling ganglion mother cell. Miranda is known to localize
Prospero protein to the basal cell cortex of neuroblasts
while the Staufen RNA-binding protein mediates Prospero
mRNA localization. miranda is shown to be required
for asymmetric Staufen localization in neuroblasts.
Miranda thus acts to partition both
Prospero protein and mRNA. Furthermore, Miranda
localizes Prospero and Staufen to the basolateral cortex in
dividing epithelial cells, which express the three proteins
prior to neurogenesis.
Analyses using miranda mutants reveal that Prospero and
Staufen interact with Miranda under the same cell-cycle-dependent
control. The wild-type Mira protein localizes predominantly to the cortex in
interphase NBs, especially to the apical cortex along with Pros
at late interphase. At the onset of prophase,
the majority of the wild-type Mira becomes localized to the
basal cortex as a crescent, while a fraction of the protein
distributes to the apical region in a punctate manner. As the mitotic stage
proceeds, an increasing proportion of Mira appears to be
incorporated in the basal crescent. While some
Mira protein is still observed apically during anaphase, most
Mira protein segregates to the basally budding GMC. This
pattern of subcellular localization is equally evident using
polyclonal and monoclonal antibodies against a C-terminal
polypeptide. mira mutations define three distinct functional regions along
the mira sequence. The N-terminal 290 amino acids region acts in the
basal localization of mira at mitosis in the NB and the epithelial cell.
The region between amino acid 447 and 727 includes a domain
necessary for the binding with Pros as well as the domain(s) required
for the asymmetric localization of Stau in the NB. The C-terminal 103
amino acids region confers the cell cycle dependence on the
interaction with Pros/Stau; the absence of this region results in the
prolonged association with Pros/Stau during interphase without rapid
proteolytic degradation in the GMC and NB. These observations suggest that the
epithelial cell and neuroblast (both of epithelial origin)
share the same molecular machinery for creating cellular
asymmetry (Matsuzaki, 1998).

An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. The Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. It is concluded that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation (Lee, 2006).

The translational repressor Brat directly interacts with the Miranda central domain and is a Miranda cargo protein specifically partitioned into the GMC daughter cell during neuroblast asymmetric cell division. Brat is the first Miranda cargo protein identified since the original finding that Prospero and Staufen were shown to be Miranda cargo proteins over 8 years ago. Prospero is a homeodomain transcriptional repressor, and Staufen is an RNA binding protein that interacts with prospero mRNA. It is unknown whether Miranda has other cargo proteins in addition to Brat, Prospero, and Staufen, and it is unclear whether all three known cargo proteins can associate with a single Miranda protein (Lee, 2006).

It is unknown how Brat promotes Prospero basal localization. A model is favored in which Brat protein stabilizes Prospero/Miranda interactions, so that Prospero protein is cytoplasmic in the absence of Brat. An obviously elevated level of cytoplasmic Prospero is not seen in brat mutant neuroblasts, but delocalization of Prospero protein from the basal crescent might not be visible over background. Alternatively, brat mutant neuroblasts may fail to transcribe or translate prospero in neuroblasts. This would most likely be an indirect effect, since Brat has been shown to only have translational repressor function. It has not been possible to detect prospero mRNA in wild-type larval neuroblasts, despite robust levels in GMCs, so this possibility has not been tested (Lee, 2006).

brat mutant brains show a dramatic increase in the number of large, proliferating Dpn+ neuroblasts between 48 and 96 hr ALH. Where do these hundreds of extra neuroblasts come from? They are unlikely to come from outside the brain, or from dedifferentiation of neurons or glia, although these models can't formally be ruled out. They are likely to derive from the pool of Dpn+ neuroblasts in the brain, because these are the primary pool of proliferating cells in the larval central brain, and thus the best candidates to generate the thousands of extra cells found in the hypertrophied brat mutant brains (Lee, 2006).

A model is proposed in which a subset of brat mutant “GMCs” enlarge into proliferative neuroblasts. This model is supported by several lines of evidence. (1) brat mutant GMCs maintain neuroblast-specific gene expression (Dpn, Miranda, Worniu); (2) brat mutants show an inverse relationship between increasing neuroblast number and decreasing neuronal number over time, consistent with GMCs forming neuroblasts instead of neurons; (3) brat mutant GMCs can be labeled by a BrdU pulse at their birth, yet most lose BrdU incorporation during the chase interval, showing that they either reenter the cell cycle or undergo cell death, and that cell death is not consistent with the brain overgrowth phenotype; (4) brat mutant telophase profiles show that all GMCs are born as small Miranda+ cells, ruling out physically or molecularly symmetric neuroblast divisions as a mechanism for increasing the neuroblast population; and (5) brat mutants show cell enlargement in other tissues, and a similar cell growth phenotype has been observed in mutants in the C. elegans brat ortholog (Lee, 2006).

What is the cellular origin of the brat mutant phenotype? brat mutant GMCs are compromised in three ways: they lack Brat translational repression activity, lack Prospero, and some may have ectopic aPKC. Loss of Brat translational repression activity could well play a role in the ectopic neuroblast self-renewal phenotype, because all brat mutants disrupting the NHL translational repression domain exhibit a brain tumor phenotype, and Brat has been previously shown to negatively regulate cell growth. Loss of Prospero also plays a role in the brat phenotype: prospero mutant GMCs have a failure to downregulate neuroblast gene expression and a failure in neuronal differentiation, similar to brat mutants. prospero null mutant embryos also show a slight delay in neuronal differentiation, although they appear to undergo normal neuroblast self-renewal. Finally, ectopic aPKC can also mimic aspects of the brat phenotype, including formation of supernumerary large Dpn+ neuroblasts. Interestingly, the mammalian paralogs of Drosophila aPKC (aPKCλ/ζ) are expressed in neural progenitors of the ventricular zone, and the mammalian Prospero ortholog Prox1 is expressed in differentiating neurons of the subventricular zone. Thus, identifying Prospero transcriptional targets and aPKC phosphorylation targets may provide further insight into the molecular mechanism of neural stem cell self-renewal in both Drosophila and mammals (Lee, 2006).

The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. This study shows that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. These results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization (Irion, 2006).

Asymmetric localization of mRNAs is a common mechanism for targeting proteins to the regions of the cell where they are required. This process is particularly important in the developing oocytes of many organisms, where localized mRNAs function as cytoplasmic determinants. This has been best characterized in Drosophila, where the localization of bicoid (bcd) and oskar (osk) mRNAs to the anterior and posterior poles of the oocyte defines the primary axis of the embryo. bcd mRNA is translated after fertilization to produce a morphogen that patterns the head and thorax of the embryo, whereas osk mRNA is translated when it reaches the posterior of the oocyte, where Oskar protein nucleates the assembly of the pole plasm, which contains the abdominal determinant nanos mRNA, as well as the germ line determinants. Localized mRNAs can also function as determinants during asymmetric cell divisions. For example, the asymmetric inheritance of mating type switching in budding yeast is controlled by the localization of Ash1 mRNA to the bud tip, which segregates the repressor ASH1p into only the daughter cell at mitosis. Similarly, prospero (pros) mRNA localizes to the basal side of Drosophila embryonic neuroblasts and is inherited by only the smaller daughter cell of this asymmetric cell division, where Prospero protein acts as a determinant of ganglion mother cell fate (Irion, 2006).

To be localized, an mRNA must contain cis-acting localization elements that are recognized by RNA-binding proteins, which couple the mRNA to the localization machinery. This process is only well understood for ASH1 mRNA, which contains four localization elements that are recognized by She3p, which then links the mRNA to the myosin motor complex Myo4p/She2p so that it can be transported along actin cables to the bud tip. Biochemical and genetic approaches have led to the identification of a number of RNA-binding proteins that associate with localized mRNAs in higher eukaryotes, but it is not known how these interactions target the mRNA to the correct region of the cell (Irion, 2006).

One of the best candidates for an RNA-binding protein that plays a direct role in mRNA localization is the dsRNA-binding protein Staufen (Stau). Staufen was first identified because it is required for the localization of osk mRNA to the posterior of the oocyte and co-localizes with it at the posterior pole. This localization depends on the polarized microtubule cytoskeleton and the plus end-directed microtubule motor kinesin, suggesting that Staufen may play a role in coupling osk mRNA to kinesin, which then transports the osk mRNA complex along microtubules. The posterior localization of osk mRNA also requires the exon junction complex components Mago nashi (Mago), Y14, eIF4AIII and Barentsz (Btz), as well as HRP48, which is needed for the formation of Staufen/osk mRNA particles (Irion, 2006).

Staufen homologues seem to play a similar role in the microtubule-dependent localization in vertebrates. GFP-Stau particles have been observed to move along microtubules in cultured neurons, and the protein is a component of large ribonucleo-protein complexes that contain kinesin and dendritically localized mRNAs. In addition, a Xenopus Staufen homologue associates with Vg1 mRNA and is required for its microtubule-dependent localization to the vegetal pole of the oocyte, which is also thought to be mediated by a kinesin (Irion, 2006).

As well as this possible conserved role in kinesin-dependent transport, Drosophila Staufen is also required for the last phase of bcd mRNA localization and co-localizes with the mRNA at the anterior of the oocyte from stage 10B onwards. Furthermore, when the bcd 3′ UTR is injected into the early embryo, it recruits Staufen into particles that move in a microtubule-dependent manner to the poles of the mitotic spindles, consistent with minus end-directed microtubule transport (Irion, 2006).

Staufen also binds to prospero mRNA and is required for its localization to the basal side of the embryonic neuroblasts. In contrast to the other examples of Staufen-dependent mRNA localization, this process depends on the actin cytoskeleton and the adapter protein Miranda (Mira) (Irion, 2006).

The varied functions of Staufen raise the question of how the same protein can function in both actin- and microtubule-dependent mRNA localization, as well as in the targeting of osk and bcd mRNAs to opposite ends of the same cell. Some insight into this comes from the analysis of Staufen protein, which contains five conserved dsRNA-binding domains (dsRBDs). In all Staufen homologues, dsRBD2 is split by a proline-rich insertion in one of the RNA-binding loops, and deletion of this insertion disrupts the localization of osk mRNA, but not that of prospero mRNA, leading to the proposal that this domain couples Staufen/mRNA complexes to a kinesin-dependent posterior localization pathway. In contrast, removal of dsRBD5 prevents the localization of prospero mRNA, whereas osk mRNA localizes normally but is not translated at the posterior of the oocyte. Indeed, dsRBD5 binds directly to Miranda to couple Staufen/prospero mRNA complexes to the actin-based localization pathway. The localization of bcd mRNA also requires dsRBD5, although the loss of the insert in dsRBD2 also affects its localization slightly (Irion, 2006).

The results above suggest that different domains of Staufen couple mRNAs to distinct localization pathways, raising the possibility that the fate of Staufen mRNA complexes may depend on which Staufen-interacting proteins are present in the cell. To test this hypothesis, the effects of expressing Miranda during oogenesis were examined to determine whether it can influence the localization of bcd or osk mRNAs (Irion, 2006).

Although Miranda is not required during oogenesis, its ectopic expression causes a striking defect in anterior–posterior axis formation that reveals several important features of the mechanisms that control the targeting and translation of localized mRNAs. Firstly, these results provide strong support for the idea that the destination of Staufen/mRNA complexes is determined by the Stau-interacting factors that are present in the cell. During wild type oogenesis, Staufen associates with osk mRNA to mediate its kinesin-dependent localization to the posterior of the oocyte at stage 9, and this requires the insertion in Staufen dsRBD2, suggesting that this domain couples Staufen/osk mRNA complexes to the posterior localization pathway. However, the expression of Miranda is sufficient to target a proportion of these complexes to the anterior. This localization is mediated through the binding of Miranda to dsRBD5 of Staufen because deletion of this domain abolishes anterior localization without affecting the transport to the posterior pole. By contrast, deletion of the insert in dsRBD2 in the presence of Miranda results in the localization of all Staufen/osk mRNA complexes to the anterior pole. Thus, these two pathways act through different domains of Staufen to direct localization to opposite ends of the same cell. These pathways compete with each other, resulting in the partitioning of the Miranda/Staufen/osk mRNA complexes to the anterior and posterior poles, but each is capable of localizing all of the complexes when the other pathway is compromised. exu and swa mutants abolish the Miranda-dependent anterior localization, and osk mRNA now localizes exclusively to the posterior, whereas btz, mago and TmII mutants block the posterior localization pathway, resulting in the localization of all osk mRNA at the anterior cortex and the formation of reverse polarity embryos (Irion, 2006).

Since dsRBD5, which is not an RNA-binding domain, is necessary and sufficient for the interaction of Staufen with Miranda, the anterior localization of osk mRNA by Miranda provides a simple in vivo assay for the binding of Staufen to osk mRNA. This reveals that neither the insert of dsRBD2 nor the RNA-binding residues of dsRBD3 are required for the stable association of Staufen with the RNA. The lack of a requirement for the insert in dsRBD2 is consistent with the observation that dsRBD2Δloop binds dsRNA in vitro when expressed on its own, whereas the full-length dsRBD2 does not. It is more surprising, however, that the mutations in dsRBD3 have no effect on Staufen binding to osk mRNA since this domain binds to dsRNA with the highest affinity in vitro, and these mutations in the five key amino acids that contact the RNA abolish the domain's RNA-binding activity in vitro. The two other functional dsRNA-binding domains in Staufen (dsRBD1 and 4) must therefore be sufficient to form a stable complex with osk mRNA (Irion, 2006).

The specific effect of a quintuple mutant in dsRBD3 on posterior localization, but not on RNA binding of full-length Staufen, further suggests that these five amino acids play a role in coupling Staufen/osk mRNA complexes to the posterior localization pathway. Although it is possible that these residues are required for an interaction with a trans-acting factor, it seems more likely that it is the association of dsRBD3 with the RNA that is important because this affects either the folding of the RNA or the conformation of Staufen protein. For example, it has been suggested that the binding of Staufen dsRBDs1, 3 and 4 to osk mRNA presents a double-stranded region of the RNA to dsRBD2, which induces a conformational change in dsRBD2 that brings together the two RNA-binding regions of the domain and loops out the large insertion, which is then exposed to interact with the transport machinery. The effect of the point mutations in dRBD3 is consistent with this model and the idea that dsRBD2 functions as an RNA-binding sensor that couples Staufen/osk mRNA complexes to factors that target it to the posterior (Irion, 2006).

Although all mRNAs that accumulate in the oocyte localize at least transiently to the anterior, several lines of evidence indicate that Miranda links Staufen and osk mRNA specifically to the bcd localization pathway. (1) All other anterior mRNAs, except bcd and hu li tai shao (hts), localize to the anterior only during stages 9–10A and become delocalized at stage 10B when rapid cytoplasmic streaming begins. In contrast, Miranda maintains osk mRNA at the anterior throughout oogenesis, so that it is still localized in a tight anterior cap in the freshly laid egg. (2) Miranda, Staufen and oskar undergo the same change in their anterior localization at stage 10B as bcd mRNA: they initially localize as a ring around the anterior cortex and then move towards the middle of the anterior when the centripetal follicle cells start to migrate inwards. (3) Like bcd, the anterior localization of osk mRNA by Miranda requires Exu, Swallow and Staufen, whereas hts mRNA localization is independent of Exu and Staufen. Since the anterior localization does not require bcd mRNA itself, Miranda cannot simply hitchhike on the bcd mRNA localization complex, and it therefore presumably links osk mRNA to the same microtubule-dependent anterior transport pathway used by bcd mRNA (Irion, 2006).

In addition to its role in osk mRNA localization, Staufen associates with bcd mRNA during the late stages of oogenesis to mediate the final steps in its localization to the anterior cortex of the oocyte. Since this localization requires the Miranda-binding domain of Staufen and Miranda couples Staufen/mRNA complexes to the bcd localization pathway, it is attractive to propose that Miranda normally mediates the late anterior localization of bcd mRNA. mira mutants have no phenotype during oogenesis, however, although the protein is expressed in late oocytes. Thus, if Miranda does play a role in bcd mRNA localization, it must function redundantly with another unidentified factor. This is perhaps to be expected given the previous evidence for redundancy in the localization of bcd mRNA. For example, none of the small deletions within the bicoid localization signal abolishes its anterior localization, indicating that it contains redundant localization elements, and two distinct bcd mRNA recognition complexes have been purified biochemically from ovarian extracts (Irion, 2006).

The elucidation of the role of Miranda in bicoid mRNA localization will require the identification of other factors that couple Staufen/bicoid mRNA complexes to the anterior localization pathway, which may function redundantly with Miranda. There are no obvious candidates for these factors, however, since Staufen is the only known protein that is specifically required for the final step of bicoid mRNA localization. Indeed, one reason why such factors may have been missed in genetic screens for mutants that disrupt bicoid mRNA localization is because they are redundant with Miranda and have no phenotype on their own. For these reasons, it is hard to address the question of redundancy using a genetic approach, but further analysis of how Miranda targets Staufen/mRNA complexes to the anterior may help resolve this issue. For example, mapping the Miranda domains that direct anterior localization may provide a clue as to the molecular nature of the unidentified factors that also fulfil this function, while screens for proteins that interact with this domain could identify other components of the anterior localization pathway (Irion, 2006).

These results reveal that Miranda, like Staufen, has the capacity to mediate both microtubule- and actin-dependent localization, raising the question whether the former plays any role in its well-characterized function during the asymmetric divisions of the embryonic neuroblasts. The localization of Miranda to the basal side of the neuroblast is actin-dependent. However, the protein also accumulates at the apical centrosome during both embryonic and larval neuroblast divisions, and this localization is even more prominent in l(2)gl or dlg mutants. Furthermore, Miranda was independently identified as a component of the pericentriolar matrix and co-localizes with γ-tubulin on all of the centrosomes at syncytial blastoderm stage. Although the centrosomes disappear in the female germ line, the anterior cortex is the major site for microtubule nucleation and γ-tubulin localization in the oocyte. Thus, Miranda may localize to the anterior of the oocyte by the same mechanism as it localizes to centrosomes (Irion, 2006).

The phenotype of mira-GFP also provides insights into the translational control of osk mRNA. In wild type ovaries, osk mRNA is translationally repressed before it is localized, and this repression is then specifically relieved once the mRNA reaches the posterior pole. In principle, translational activation of osk mRNA could occur by a specific signal at the posterior, but it could also be due to some other consequence of localization, such as the concentration of the RNA in a small region or its association with the oocyte cortex. Evidence in favor of a specific posterior signal comes from an experiment in which a LacZ reporter gene under the control of the oskar 5′ region and the first 370 nt of the 3′ UTR was targeted to the anterior by the bcd localization element. Since this anterior RNA was not translated, concentration at the cortex appeared to be insufficient to relieve BRE mediated repression. However, it has recently emerged that this reporter RNA lacks the two clusters of insulin growth factor II mRNA-binding protein (IMP) binding elements in the distal oskar 3′ UTR that are essential for oskar translational activation at the posterior, making it hard to draw any conclusions from the lack of translation of this reporter RNA at the anterior. Mira-GFP provides an alternative way to test this hypothesis because it directs the anterior localization of wild type osk mRNA, with all of its translational control elements intact. This anterior mRNA is not translated during stages 9–13, despite being efficiently localized to the cortex, whereas the osk mRNA at the posterior of the same oocytes is translated normally. Thus, concentration at the cortex is not sufficient to de-repress translation, strongly supporting the idea that activation depends on a specific posterior signal (Irion, 2006).

Although the anterior osk mRNA is not translated at the normal time, the repression system breaks down at the very end of oogenesis, and the mRNA is very efficiently translated in mature oocytes. This suggests that some key component of the repression system disappears at this stage, and a good candidate is the BRE-binding protein Bruno. Bruno is highly expressed during oogenesis but is not detectable in embryos. Furthermore, the addition of Bruno is sufficient to cause the repression of exogenous osk mRNA in an embryonic translation system. These results indicate that Bruno is degraded at the end of oogenesis, whereas all other components necessary for translational repression of osk mRNA are still present in the embryo. Thus, the translation of anterior osk mRNA in mira-GFP oocytes is most probably triggered by the disappearance of Bruno (Irion, 2006).

Once it is translated at the posterior of the oocyte, Oskar protein nucleates the formation of the pole plasm with its characteristic electron-dense polar granules, which gradually assemble during stages 9–14 of oogenesis. This appears to be a stepwise process, in which Oskar protein recruits some polar granule components as soon as it is translated at stage 9, such as Vasa and Fat facets, while other components are added in sequence during the rest of oogenesis. For example, Tudor, Capsuleen and Valois are recruited during stage 10A, whereas nanos, Pgc and gcl mRNAs only become enriched at the posterior at stages 10B–11. It is therefore surprising that the anterior Oskar protein, which is only synthesized in stage 14 oocytes, can still nucleate fully functional pole plasm that induces the formation of anterior pole cells. Thus, although the pole plasm normally assembles in an ordered fashion over the last 5 stages of oogenesis, this whole process can still occur once oogenesis is complete. This indicates that the assembly of the pole plasm does not depend on the order of addition of its components, all of which must still be present and freely diffusible in mature oocytes (Irion, 2006).

Asymmetric localization of cell fate determinants is a crucial step in neuroblast asymmetric divisions. Whereas several protein kinases have been shown to mediate this process, no protein phosphatase has so far been implicated. In a clonal screen of larval neuroblasts, the evolutionarily conserved Protein Phosphatase 4 (PP4) regulatory subunit PP4R3/Falafel (Flfl) was identified as a key mediator specific for the localization of Miranda (Mira) and associated cell fate determinants during both interphase and mitosis. Flfl is predominantly nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown. Analyses of nuclear excluded as well as membrane targeted versions of the protein suggest that the asymmetric cortical localization of Mira and its associated proteins during mitosis depends on cytoplasmic/membrane-associated Flfl, whereas nuclear Flfl is required to exclude the cell fate determinant Prospero (Pros), and consequently Mira, from the nucleus during interphase/prophase. Attenuating the function of either the catalytic subunit of PP4 (PP4C; Pp4-19C in Drosophila) or of another regulatory subunit, PP4R2 (PPP4R2r in Drosophila), leads to similar defects in the localization of Mira and associated proteins. Flfl is capable of directly interacting with Mira, and genetic analyses indicate that flfl acts in parallel to or downstream from the tumor suppressor lethal (2) giant larvae (lgl). These findings suggest that Flfl may target PP4 to the MIra protein complex to facilitate dephosphorylation step(s) crucial for its cortical association/asymmetric localization (Sousa-Nunes, 2009).

Drosophila neuroblasts (NBs) are stem-cell-like neural progenitors, which undergo repeated asymmetric divisions to self-renew and generate neurons and/or glia. During each round of division the cell fate determinants Pros (a homeodomain-containing transcription regulator), Numb (a negative regulator of Notch signaling), as well as Brain Tumor (Brat, whose mechanism of action in cell fate specification is unclear) are asymmetrically localized as protein crescents on the NB cortex. In the embryo, the NB mitotic spindle is oriented along the apicobasal axis, the cell fate determinants and their adapter proteins localize to the NB basal cortex and segregate exclusively to the smaller basal daughter, called ganglion mother cell (GMC). The GMC divides terminally to produce two neurons or glial cells. The coordination between the basal localization of the cell fate determinants and the apicobasal orientation of the spindle during mitosis is mediated by several evolutionarily conserved proteins that localize to the apical NB cortex during the G2 stage of the cell cycle. These comprise [1] the Drosophila homologs of the Par3/Par6/aPKC protein cassette, [2] several proteins involved in heterotrimeric G protein signaling—Gαi/Partner of Inscuteable (Pins)/Locomotion defects (Loco), [3] as well as Inscuteable (Insc). In contrast to the embryo, NBs in the larval central brain divide without an apparent fixed orientation. Nevertheless the majority of central brain NBs appear to utilize the same molecular machinery as embryonic NBs, with the apical and basal molecules sharing similar hierarchical relationships and localizing to opposite sides of the NB cortex (Sousa-Nunes, 2009).

Asymmetric localization of Pros and Brat on the one hand and Numb on the other, is mediated through direct interactions with their respective adapters, the coil-coil proteins Miranda (Mira) and Partner of Numb (Pon). Although mutations affecting any of the apical proteins compromise asymmetric localization of basal proteins to varying extents, only in the case of aPKC has any mechanistic insight emerged. aPKC facilitates basal localization of cell fate determinants either through phosphorylation of the cytoskeletal protein Lgl and/or through direct phosphorylation of the determinant. Lgl is uniformly localized throughout the NB cortex, and is essential for cortical association and asymmetric localization of the cell fate determinants and their adapters. aPKC phosphorylates Lgl on three conserved serine residues and the triphosphorylated form appears to be inactive due to a conformational change. The proposed model is that unphosphorylated, active Lgl is restricted to the basal cortex because of apically localized aPKC. Consistent with this model, a nonphosphorylatable version of Lgl, Lgl3A, in which the three target serines have been mutated to alanines, appears to be constitutively active and its expression leads to uniform cortical localization of the normally basally restricted cell fate determinants. Numb is a second protein that can be phosphorylated by aPKC and phosphorylation of three N-terminal serines causes it to become cytoplasmic (Sousa-Nunes, 2009).

How Lgl acts to facilitate the localization of cell fate determinants is less clear. Lgl can bind nonmuscle Myosin II (Zipper) and genetic experiments suggest that Myosin II and Lgl have antagonistic activities. Hence, one possible scenario would be that Myosin II is active at the apical cortex due to the presence of phosphorylated Lgl, which is incapable of binding to Myosin II. Myosin II can then act to exclude basal proteins from the apical cortex. Alternatively, since yeast Lgl orthologs function in exocytosis, it has been suggested that Lgl might act by regulating this process. It is possible that Lgl positively promotes delivery and cortical association of the basal molecules, and that this is antagonized by Myosin II apically. In this scenario, Lgl is inhibited apically both by aPKC and Myosin II, and only basal Lgl is active and able to promote cortical association of the basal proteins (Sousa-Nunes, 2009).

The unconventional Myosin VI (Jaguar, Jar) and Myosin II bind in a mutually exclusive manner to the basal adapter protein Mira. However, in contrast to Myosin II, which acts antagonistically to Lgl, Jar acts in a synergistical manner with Lgl to effect Mira basal localization. In mitotic NBs devoid of Jar, Mira is mislocalized to the cytoplasm. Jar possibly mediates association of Mira with the basal actin cytoskeleton (Sousa-Nunes, 2009).

In addition to aPKC, a few other serine/threonine protein kinases have been shown to play a role in facilitating asymmetric protein localization in NBs. These include Cdk1, required for the asymmetric localization of both apical and basal components during mitosis, Aurora A (AurA), and Polo, both of which mediate Numb and Pon asymmetric localization. With the exception of Polo kinase, which phosphorylates a serine residue within the Pon asymmetric localization domain, substrates for the other kinases have not been identified. The involvement of protein kinases in NB asymmetric divisions implies the involvement of protein phosphatases; however, to date, none have been implicated in the process (Sousa-Nunes, 2009).

In a clonal genetic screen designed to identify genes that mediate NB asymmetric divisions, multiple loss-of-function alleles of flfl. Falafel (Flfl) were identified as a regulatory subunit of the evolutionarily conserved Protein Phosphatase 4 (PP4) Phosphatase complex. PP4 belongs to the best-studied family of cellular protein serine/threonine phosphatases, PP2A (the other major families being PP1, PP2B, and PP2C). Similarly to other PP2A-like phosphatases, PP4 functions as a heterotrimeric complex comprising of a catalytic subunit, PP4C, associated with two regulatory subunits, PP4R2 and PP4R3. PP4, or specifically PP4R3/Flfl, has been implicated in a variety of molecular and cellular processes including regulation of MEK/Erk, insulin receptor substrate 4, Hematopoietic progenitor kinase 1, and Histone deacetylase 3 activities, centrosome maturation, cell cycle progression, apoptosis, DNA repair, cell morphology, and lifespan control (Sousa-Nunes, 2009 and references therein).

This study shows that loss of flfl, as well as attenuation of PP4C/Pp4-19C or PPR2/PPp4R2r function by RNAi specifically results in delocalization of Mira and its associated proteins throughout the cytoplasm in metaphase/anaphase NBs; in addition, both Mira and Pros localize to the NB nucleus prior to nuclear envelope breakdown. Excessive nuclear Mira is dependent on the presence of Pros. These results suggest that whereas cytoplasmic or membrane-associated PP4 is required for asymmetric cortical localization of Mira (and its associated proteins) during metaphase and anaphase, nuclear PP4 is required to exclude Pros (and as a consequence, Mira) from the NB nucleus prior to nuclear envelope breakdown. Moreover, Flfl can complex with Mira in vivo and directly interact with Mira, suggesting that Flfl targets PP4 activity to the Mira complex to facilitate its correct localization (Sousa-Nunes, 2009).

In a clonal screen on third-instar larval (L3) brains, designed to identify novel genes on chromosome arm 3R required for NB asymmetric division, a novel allele of flfl, flfl795 was isolated. In metaphase and anaphase flfl795 clone NBs, Mira displays weak cortical crescents but also a pronounced mislocalization throughout the cytoplasm, whereas in surrounding heterozygous NBs Mira is localized to a robust crescent like in wild type with little cytoplasmic accumulation. As with many mutations that disrupt NB asymmetry during metaphase and anaphase, flfl795 NBs display telophase rescue: The majority of the cytoplasmic Mira relocalizes asymmetrically to the NB cortex at telophase, resulting in asymmetric segregation of Mira into the GMC. Using the flfl795 allele, two additional alleles [flfl795(2), flfl795(3)] were identified via complementation screening of an independent collection of ethylmethane sulfonate (EMS) mutant stocks. Sequencing of these three EMS-induced flfl alleles revealed single point mutations resulting in premature stop codons at positions 324 (flfl795) and 630 [flfl795(2)] of the longest isoform (980 amino acids) and a disruption to the splice acceptor site at the 3′ end of the fourth intron [flfl795(3)]. All three alleles display a mislocalization of Mira to the cytoplasm of metaphase NBs and form an allelic series in terms of phenotypic severity: flfl795 > flfl795(3) > flfl795(2) (Sousa-Nunes, 2009).

Homozygous flfl795 animals survive to pharate adults whereas hemizygous flfl795 animals [using Df(3R)Exel6170 to remove one copy of the flfl coding region] only survive until L3. Furthermore, although the cytoplasmic Mira phenotype of flfl795 homozygotes is highly penetrant, the majority of metaphase NBs still display weak Mira crescents, whereas the majority of metaphase NBs of flfl795 hemizygotes display no crescents. These results suggest that the strongest EMS allele (flfl795) is nevertheless a hypomorph. Therefore a flfl-null allele (flflN42) was generated by imprecise excision of the P-element P{EPgy2}flflEY03585, located ∼1 kb upstream of the flfl translational start site. This allele was confirmed to be a genetic null by the similar expressivities of NB phenotypes in flflN42 homozygotes and flflN42 hemizygotes, as well as in flfl795/flflN42 and flfl795/Df(3R)Exel6170. Consistently, flflN42 NBs are antigen-minus (see below) and molecular analysis indicates that it is a deletion extending into the coding region, deleting the first 1075 base pairs of the coding sequence. Subsequent analyses of the phenotype were carried out using the flflN42 allele, hereafter referred to simply as flfl (Sousa-Nunes, 2009).

In addition to the mislocalization of Mira, the Mira-associated proteins Pros, Brat and Staufen (Stau), are similarly mislocalized to the cytoplasm of metaphase/anaphase flfl NBs. Pros mislocalization occurs in Asense (Ase)-positive NB lineages which comprise the majority of lineages in the central brain; Ase-negative NBs are Pros-negative in flfl as well as in wild-type brains. In contrast, the localization of members of the other basal complex, Pon and Numb, and of apical complexes is unaffected. Hence, during NB division, flfl loss of function specifically affects the localization of the Mira complex (Sousa-Nunes, 2009).

Flfl homologs have been identified in several species, from yeast to humans. They all possess the same domain architecture: a Ran-binding domain (RanBD) at the N terminus, similar in three-dimensional structure to the Ena/VASP homology domain 1 (EVH1, which derives its name from the founding members Enabled and Vasodilator-stimulated phosphoprotein) and to the pleckstrin homology domain; followed by a conserved domain of unknown function (DUF625), a region containing armadillo/HEAT repeats, and a region of low complexity. Within the DUF625 domain, Flfl contains two putative NLSs (NLS1 and NLS2) as well as a nuclear export signal (NES); close to the C terminus Flfl contains a short conserved stretch of acidic and basic amino acid residues that has been shown to be required for nuclear localization of the Dictyostelium discoideum homolog, SMEK (NLS3). Flfl contains many putative target sites for O-linked N-acetylglucosamine (O-GlcNAc) glycosylation in its C-terminal 300 amino acids and numerous putative phosphorylation sites throughout, some of which are predicted to be PKC targets (Sousa-Nunes, 2009).

In conclusion, loss of function or RNAi knockdown of the regulatory subunits flfl/PP4R3 or PPP4R2r/PP4R2 as well as knockdown of the catalytic subunit Pp4C-19C/PP4C of PP4 causes mislocalization of Mira/Pros/Brat/Stau to the cytoplasm of metaphase and anaphase NBs (Sousa-Nunes, 2009).

Attenuation of PP4 function above also causes increased frequency of nuclear Mira/Pros prior to nuclear envelope breakdown. The observation that depletion of the catalytic subunit of PP4 results in identical phenotypes to the depletion of its regulatory subunits, suggests that phosphatase activity plays a role in the localization of Mira/Pros throughout the NB cell cycle (Sousa-Nunes, 2009).

Nuclear mislocalization of Mira seen in flfl, jar, or mira2L150 single-mutant NBs requires pros function. This suggests that, when transport of Mira toward or its tethering to the cortex is defective, Pros can take Mira into the nucleus. In this context, the normal relationship between Mira and Pros is reversed, with Pros instructing Mira localization rather than the converse. In the absence of pros, Mira is not localized to the nucleus, even when PP4 function is attenuated. Thus, the role of PP4 on these two temporally distinct localizations of Mira/Pros appears to involve distinct targets since one is a Mira-dependent localization and the other is Pros-dependent (Sousa-Nunes, 2009).

In contrast to serine-threonine kinases, substrate specificity for serine/threonine protein phosphatases is thought to be conferred not primarily by sequences adjacent to the target residues but rather by interaction between the substrate and regulatory subunits of the phosphatase complex. This is the case for the founding family member PP2A, whose variable subunit composition can also target the complex to distinct subcellular domains and is thought to be the case also for PP4. Flfl, a regulatory subunit of PP4, is able to bind Mira and Flfl and Mira are found in a complex in vivo. No binding was detected between Flfl and Pros but since Mira and Pros still colocalize when PP4 function is attenuated, these results also suggest that PP4 function is not required for the Mira-Pros interaction. Therefore, Pros could be recruited to PP4 by its association with Mira, which in turn binds Flfl (Sousa-Nunes, 2009).

Flfl is nuclear before and cytoplasmic after nuclear envelope breakdown. The results from nuclear excluded and membrane targeted versions of Flfl suggest that nuclear Flfl is required to exclude Mira/Pros from the nucleus when inefficiently bound to the cytoskeleton/cortex, whereas cytosolic or membrane-associated Flfl is required for the cortical association and asymmetric localization of Mira/Pros/Brat/Stau at metaphase and anaphase. The localization of Mira/Pros prior to and after nuclear envelope breakdown by PP4 may involve different phosphatase substrates. It is tempting to entertain the possibility that Mira dephosphorylation by PP4 in the cytoplasm is required for its asymmetric cortical localization during mitosis, and that Pros dephosphorylation by PP4 in the nucleus is required for its nuclear exclusion/progression through prometaphase. Indeed, a previous study has shown that cortical Pros is highly phosphorylated relative to nuclear Pros. To test this hypothesis, attempts were made to detect enrichment of a lower mobility band of Mira::3GFP in flfl larval extracts compared with wild type but this it could not be detected, working at the limits of detectability (Sousa-Nunes, 2009).

Asymmetric cortical localization of proteins during NB asymmetric division is dependent on an intact actin cytoskeleton. Although flfl is required for Mira cortical association, at no point in the NB cell cycle does Flfl exhibit cortical enrichment. However, modified versions of Flfl that are either uniformly cytoplasmic or cortically enriched can both drive asymmetric cortical localization of Mira and its associated proteins. Moreover, the Mira mislocalization phenotypes of flfl are strikingly similar to those of Myo VI/jar. Both mutants exhibit nuclear Mira/Pros prior to and cytoplasmic Mira and associated proteins following NB nuclear envelope breakdown; both Flfl and Jar are cytoplasmic at metaphase/anaphase; and genetically, both Jar and Flfl act parallel to or downstream from Lgl. Further propelled by the presence of a putative actin-binding domain in Flfl (the RanBD domain, which is an EVH1-like domain), it was asked whether Flfl too might facilitate association of Miranda with the actin cytoskeleton either separately from or in association with Jar. However, in vitro assays clearly showed that Flfl does not bind F-actin, although Mira alone does, with comparable strength to that of α-Actinin and Jar, used as controls. Furthermore, Jar could not be detected in Flfl containing protein immunoprecipitates. Therefore, it seems unlikely that Flfl acts either directly or in a complex with Jar to facilitate Mira transport along or tethering to the actin cytoskeleton. Still, Flfl could act indirectly; for example, by stabilization of the Mira-Jar association. It is speculated that Flfl may act by targeting PP4 to the Mira complex and that the consequent dephosphorylation of a component of this complex facilitates Jar-Mira association (Sousa-Nunes, 2009).

In Dictyostelium, mutants in the flfl homolog, smkA, exhibit phenotypes similar to strains defective in Myo II assembly, suggesting that smkA may regulate Myo II function. However, in flfl NBs the Mira mislocalization phenotype does not resemble that of Myo II loss of function, which has been described to lead to Mira mislocalization to the mitotic spindle in embryonic NBs (Sousa-Nunes, 2009).

The reduced proliferation seen in flfl NBs correlates with nuclear localization of Pros/Mira. Nuclear Pros negatively regulates transcription of cell cycle genes and positively regulates differentiation genes, and has been shown to limit NB proliferation. Therefore, ectopic nuclear Pros is likely to be at least one cause of the NB underproliferation observed in flfl brains. Still, it is possible that flfl has additional functions in promoting proliferation, independent of its role in excluding Pros/Mira from the NB nucleus. Indeed, an excessive proportion of phospho-histone H3-positive flfl NBs was detected relative to wild type. These NBs typically had a nucleus but the cell morphology was not spheroid, as would be expected in prophase cells. This suggests that flfl NBs either have a block or delay in prometaphase or that PP4 may be required for dephosphorylation of Histone H3; in either case, it seems to be required for dephosphorylation of other proteins involved in cell cycle progression. Nonetheless, pros,flfl double-mutant NB clones are indistinguishable from those of pros single mutants, both showing extensive overproliferation, suggesting that the loss of flfl is unable to override the overproliferation induced by loss of pros (Sousa-Nunes, 2009).

Asymmetric cell divisions generate daughter cells with distinct fates by polarizing fate determinants into separate cortical domains. Atypical protein kinase C (aPKC) is an evolutionarily conserved regulator of cell polarity. In Drosophila neuroblasts, apically restricted aPKC is required for segregation of neuronal differentiation factors such as Numb and Miranda to the basal cortical domain. Whereas Numb is polarized by direct aPKC phosphorylation, Miranda asymmetry is thought to occur via a complicated cascade of repressive interactions (aPKC -| Lgl -| myosin II -| Miranda). This study provides biochemical, cellular, and genetic data showing that aPKC directly phosphorylates Miranda to exclude it from the cortex and that Lgl antagonizes this activity. Miranda is phosphorylated by aPKC at several sites in its cortical localization domain and phosphorylation is necessary and sufficient for cortical displacement, suggesting that the repressive-cascade model is incorrect. In investigating key results that led to this model, it was found that Y-27632, a Rho kinase inhibitor used to implicate myosin II, efficiently inhibits aPKC. Lgl3A, a nonphosphorylatable Lgl variant used to implicate Lgl in this process, inhibits the formation of apical aPKC crescents in neuroblasts. Furthermore, Lgl directly inhibits aPKC kinase activity. It is concluded that Miranda polarization during neuroblast asymmetric cell division occurs by displacement from the apical cortex by direct aPKC phosphorylation. Rather than mediating Miranda cortical displacement, Lgl instead promotes aPKC asymmetry by regulating its activity. The role of myosin II in neuroblast polarization, if any, is unknown (Atwood, 2009).

This study examined the mechanism by which polarity is generated in Drosophila neuroblasts, a process required for the segregation of cell fate determinants during asymmetric cell division. This process utilizes aPKC, which is found in many polarized systems such as epithelia. Previously, polarization of the protein Miranda, which is normally restricted to the basal neuroblast cortex opposite aPKC, has been thought to occur by a complex cascade of repressive interactions involving the tumor suppressor Lgl and the motor protein myosin II. The finding that aPKC phosphorylation displaces Miranda from the cortex of neuroblasts and S2 cells led to the idea that the repressive-cascade model might not accurately describe Miranda displacement. This prompted a reexamination of key results supporting the repressive-cascade model (Atwood, 2009).

Based on previous results, it is proposed that studies suggesting that myosin II is involved in aPKC-mediated cortical displacement of Miranda are an artifact of inhibition of aPKC by the Rho kinase inhibitor Y-27632. Although the possibility cannot be excluded that the Miranda polarity defects observed in Y-27632-treated embryos are indeed the result of myosin II inhibition, the fact that this phenotype is identical to that exhibited by apkc mutants, the efficient inhibition of aPKC, and the high concentrations of this compound used in previous reports (~50 mM, compared to the IC50 < 10 μM for aPKC and 0.1 μM for Rho kinase) indicate that the simplest interpretation of the Y-27632 phenotype is direct inhibition of aPKC activity. The role of myosin II in Miranda cortical displacement, if any, is unclear (Atwood, 2009).

The central result that led to the placement of Lgl between aPKC and Miranda was reexamined. Expression of a form of Lgl in which the aPKC phosphorylation sites have been inactivated results in uniformly cortical Miranda in neuroblasts. This result can be interpreted in one of two ways: Lgl mediates Miranda cortical targeting and phosphorylation of Lgl represses this activity, or Lgl inhibits aPKC and this inhibition is repressed by aPKC phosphorylation (i.e., feedback). The key distinction between these two models is whether or not aPKC is repressed when Lgl3A is expressed. Several recent studies indicate that Lgl is a potent inhibitor of aPKC activity. Consistent with this, it was found that Lgl3A expression dramatically reduces the localization of aPKC to the neuroblast apical cortex. Furthermore, it was found that a form of aPKC that is not efficiently repressed by Lgl can overcome the effects of Lgl3A and drive Miranda into the cytoplasm, consistent only with Lgl phosphorylation not being a requirement for Miranda cortical displacement. In addition, it was shown that Lgl alone is sufficient for inhibition of aPKC activity. Thus, it is concluded that Lgl can directly inhibit aPKC and is not required for Miranda cortical targeting (Atwood, 2009).

A simpler mechanism than the repressive-cascade model for Miranda polarization by aPKC is favored: aPKC phosphorylates Miranda, causing it to be displaced from the cortex. The identification of Miranda as a direct aPKC substrate, the requirement of these phosphorylation events for cortical displacement in both S2 cells and neuroblasts, and the necessity of these phosphorylation events for normal development and viability support this model. The sufficiency of phosphorylation (phosphomimetic Miranda is cytoplasmic in the absence of aPKC) indicates that other phosphorylation events (such as phosphorylation of Lgl in the repressive-cascade model) are not required for Miranda cortical displacement. This new model dramatically simplifies the understanding of how asymmetric aPKC activity, as generated by Baz and Cdc42, is translated into the segregation of cell fate determinants. Thus, polarization of three components downstream of aPKC, Numb, and Miranda (this work), appears to occur by direct aPKC phosphorylation. Further work will be required to determine whether this mechanism is utilized by all factors that are polarized by aPKC (Atwood, 2009).